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Synthesis of Coumarins Linked With 1,2,3-Triazoles under
Microwave Irradiation and Evaluation of their Antimicrobial and
Antioxidant Activity
Muthipeedika Nibin Joy1,2*, Yadav D. Bodke2,3*, Sandeep Telkar4,
Vasiliy A. Bakulev5 1Innovation Center for Chemical and
Pharmaceutical Technologies, Institute of Chemical Technology, Ural
Federal University, 19 Mira Street, Yekaterinburg, 620002.
2Department of P.G studies and Research in Industrial Chemistry,
Kuvempu University, Jnana Sahyadri, Shankaraghatta, Shimoga,
Karnataka, India- 577451. 3Department of P.G studies and Research
in Chemistry, Kuvempu University, Jnana Sahyadri, Shankaraghatta,
Shimoga, Karnataka, India- 577451. 4Department of P.G studies and
Research in Biotechnology, Kuvempu University, Jnana Sahyadri,
Shankaraghatta, Shimoga, Karnataka, India- 577451. 5TOS Department,
Ural Federal University, 19 Mira Street, Yekaterinburg, 620002.
*Corresponding author: Muthipeedika Nibin Joy, email:
[email protected], Phone: +7-9634431830 Received November 17th,
2019; Accepted December 4th , 2019. DOI:
http://dx.doi.org/10.29356/jmcs.v64i1.1116 Abstract. A series of
coumarin derivatives linked with 1,2,3-triazoles has been
synthesized by utilizing the copper catalyzed azide-alkyne
cycloaddition reaction and were screened for their antimicrobial
and antioxidant properties. Some of the compounds displayed
promising antibacterial activities (MIC ranging from 5-150 µg/mL)
and moderate antifungal activities as compared to the respective
standards. The compounds 4k and 4g displayed good antibacterial
activity when compared with the standard, Ciprofloxacin, and 4n
exhibited better antifungal activity when compared to other
synthesized compounds. The in silico docking studies of the active
compounds were carried out against the gyrase enzyme and from those
studies, it was acknowledged that 4k possessed significant hydrogen
bonding and hydrophobic interactions which could be the plausible
reason for its superior activity as compared to the other
synthesized compounds. The compounds 4h and 4q showed promising
antioxidant activity when compared with the standard, BHT, which
could be attributed to the presence of electron donating
substituents. Keywords: Coumarin; 1,2,3-triazole; click chemistry;
antimicrobial; antioxidant. Resumen. Una serie de derivados de
cumarina enlazados con 1,2,3-triazoles fue sintetizada empleando la
reacción de cicloadición azida-alquino catalizada con cobre y fue
evaluada en sus propiedades antimicrobianas y antioxidantes.
Algunos de los compuestos exhibieron actividad antimicrobiana
promisoria (intervalo MIC de 5-150 µg/mL) y actividad antifúngica
moderada con respecto a los estándares respectivos. Los compuestos
4g y 4k mostraron buena actividad antibacterial con relación al
estándar. Fluconazole y 4n exhibieron mejor actividad antifúngica
en comparación con el resto de los compuestos. Se llevaron a cabo
estudios in silico de modelado molecular e interacción de los
compuestos activos con la enzima girasa ADN. De estos estudios se
observó que 4k posee enlaces puentes de hidrógeno e interacciones
hidrofóbicas significativos, los cuales podrían ser una causa
plausible de su actividad mayor a aquélla mostrada por los otros
compuestos sintetizados. Los compuestos 4h y 4q mostraron una
importante actividad antioxidante comparada con el estándar (BHT),
lo cual podría atribuirse a la presencia de sustituyentes
electro-donadores. Palabras clave: Cumarina; 1,2,3-triazol;
reacciones click; antimicrobiano; antioxidante
mailto:[email protected]://dx.doi.org/10.29356/jmcs.v64i1.1116
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Introduction
The discovery of different types of microorganisms has explained
the main reasons for various infectious diseases responsible for
the most complex health issues of this century. Organisms like
bacteria, fungi and viruses are identified to cause serious health
hazards globally which may even lead to death [1]. Although a lot
of drugs as potent antimicrobial agents have been identified
hitherto, the rise of resistant microorganisms or the development
of multi drug resistance in pathogens still remain as a major
concern worldwide [2]. Hence, the discovery of new drugs with
potent anti-microbial activity, particularly against the resistant
strains is therefore highly needed to solve this problem [3].
Highly reactive free radicals and oxygen species that are present
in the biological systems may abstract hydrogen atom from membrane,
lipid, protein, DNA etc. and consequently lead to damages of
several biological species and hence can initiate numerous
degenerative diseases [4]. The impairments caused by free radicals
can lead to aging, cancer, atherosclerosis and some other serious
disorders. Therefore, the removal of free radicals from biological
system is very important for the sustainability of cellular
machinery and for preventing the commencement and propagation of
oxidative diseases [5]. The supplementation of antioxidants (Free
radical scavengers) is found to be beneficial for avoiding
oxidative damages as they have the ability to trap free radical
species.
Coumarins are an important class of benzopyrones found in green
plants either in free or combined state and display wide spectrum
pharmacological activities [6]. Natural coumarins and their
derivatives are of great interest due to their widespread
biological properties and have attracted many medicinal chemists
for further derivatization and screening them as novel therapeutic
agents. Coumarins are reported to be active as antibacterial [7],
anti-inflammatory [8] and antiviral agents [9] and the various
therapeutic applications of coumarin derivatives include photo
chemotherapy, anti-tumor therapy and anti-HIV therapy [10]. The
coumarin motif is present within the chemical structure of
pharmaceutical drugs such as warfarin, acenocoumarol, carbochromen
etc. and in antibiotics such as novobiocin, clorobiocin and
coumermycin A1 [11,12]. In view of these interesting
pharmacological properties, the exploration of natural or synthetic
coumarin derivatives has intrigued chemists for decades for their
applicability as drugs. On the other hand, 1,2,3-triazoles are
found in diverse bioactive compounds and have shown numerous
biological potentials like anticancer [13], immunosuppressant [14],
antimicrobial [15], antiviral [16], antiallergic [17] and
anti-inflammatory activities [18]. The exceptional properties of
1,2,3-triazoles include high dipole character and hydrogen bonding
capability and hence can be used as linkers of various molecules.
Furthermore, these compounds are highly rigid and stable under
acid/base hydrolysis and oxidative/reductive conditions which makes
it a metabolically stable heterocyclic ring [19,20]. Several
1,2,3-triazole containing drug molecules are now available in the
market (Fig 1) or is in the clinical trials of final stage
[21].
Fig 1. Some of the available drugs containing 1,2,3-triazole
moiety.
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Nowadays, the microwave-assisted organic synthesis (MAOS) is
rapidly becoming recognized as a valuable tool for facilitating a
wide variety of transformations and hence has significantly
extended its scope in drug discovery laboratories [22]. It is well
documented that the microwave assistance can lead to remarkable
rate enhancement with better reproducibility and less side
reactions as compared to standard heating methodologies [23]. In
the design and development of new drugs, the employment of
molecular hybridization strategy which involves the combination of
different pharmacophores may lead to compounds with interesting
biological profiles. These combined chemical entities recognized
and derived from known bioactive molecules possessing different
mechanisms of action could be beneficial for various treatments as
they possibly offer some advantages in overcoming drug resistance
as well as improving their biological potency [24,25].
Since the combination of two pharmacophores on the same scaffold
is a well established approach to more potent drugs [24-27], we
focused our attention in the synthesis of some pharmacologically
relevant coumarin derivatives containing 1,2,3-triazole moiety.
Owing to the instability of coumarin nuclei in basic as well as
prolonged heating conditions and as a continuation of our ongoing
research in the synthesis of some biologically active molecules
[28-31], it has been planned to utilize the copper catalyzed
Huigsen 1,3 dipolar cycloaddition, commonly known as click
chemistry, for the synthesis of various coumarin analogues under
microwave irradiation. The synthesis and biological evaluation of
1,2,3-triazole derivatives linked with coumarin moiety by utilizing
copper catalyzed azide–alkyne cycloaddition is recently reported in
the literature by various research groups [32-35]. However, most of
these results were limited to the usage of aromatic azides and a
very few benzylic azides, and the reaction times are generally very
high (18-24 h) that may lead to various side-products. Moreover,
the scope of diverse aliphatic, acyclic and cyclic azides in this
area is relatively unexplored and challenging as it is difficult to
handle aliphatic azides. Furthermore, aliphatic azides are less
reactive and extremely stable in almost all the reaction media and
hence it requires prolonged reaction times for complete conversion
of starting materials to products. These observations prompted us
to optimize the planned synthetic methodology under microwave
irradiation as it will be less time consuming and highly efficient
with less side products. In this paper, we report a facile,
convenient and rapid access for the microwave irradiated synthesis
of a series of coumarins linked with 1,2,3-triazoles. The
synthesized compounds were screened for their antimicrobial and
antioxidant potencies, and the in-silico docking studies of
selected compounds against gyrase enzyme has been subsequently
investigated.
Experimental Chemistry General
All solvents and reagents were obtained from commercial
suppliers and used without any further purification unless
otherwise noted. Microwave reactions were performed in a single
mode Biotage Initiator Microwave Synthesizer and temperature was
monitored using infrared. Analytical TLC was performed on
pre-coated aluminum sheets of silica (60 F 254 nm) and visualized
by short-wave UV light at λ 254. Melting points were determined on
an EZ - Melt automated melting point apparatus. 1H NMR (400 or 300
MHz) and 13C NMR (100 or 75 MHz) were recorded on Bruker Avance II
spectrometer and chemical shifts were measured in δ (ppm). The
following abbreviations are used for the splitting patterns: s for
singlet, d for doublet, t for triplet, m for multiplet and br for
broad. LC-MS analyses were performed using ESI/APCI, with an
ATLANTIS C18 (50 X 4.6 mm-5µm) column and a flow rate of 1.2
mL/min.
Procedure for the synthesis of 4-methyl-7-hydroxy coumarin
intermediate (2)
To the weighed quantity of resorcinol (1 equiv.) and ethyl
acetoacetate (1.1 equiv.), the ionic liquid [bmim]Cl·2AlCl3 (1.1
equiv.) was added and the reaction mixture was stirred at 30 °C for
20 min. All additions were carried out in an inert atmosphere. The
reaction was quenched by adding 6 M HCl in cold conditions. The
resultant product was filtered and further purified by column
chromatography to obtain the titled compound 2 as off white solid
in 88 % yield. mp: 180-182 °C; 1H NMR (400 MHz, DMSO-d6): δ 2.34
(s, 3H, CH3), 6.10 (s, 1H, ArH), 6.69 (d, J=2.3 Hz, 1H, ArH), 6.78
(dd, J=8.7, 2.3 Hz, 1H, ArH), 7.56 (d, J=8.7 Hz, 1H, ArH), 10.48
(bs, 1H, OH). 13C NMR (100 MHz, DMSO-d6): δ 18.0, 102.1, 110.2,
112.0, 112.8, 126.5, 153.5, 154.8, 160.2,
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161.1. LC-MS: Calculated 176.2, Observed 177.2. Analysis calcd
for C10H8O3: C, 68.18, H, 4.58, O, 27.25 %, found: C, 68.21, H,
4.57, O, 27.22 %. Procedure for the synthesis of
4-methyl-7-(prop-2-ynyloxy)-2H-chromen-2-one intermediate (3)
To the weighed quantity of 4-methyl-7-hydroxy coumarin 2 (1
equiv.) in acetone, were added K2CO3 (3 equiv.) and propargyl
bromide (1.2 equiv.) in inert atmosphere and the reaction mixture
was stirred at RT for 12 hours. The reaction completion was
monitored by TLC and the mixture was poured into ice-cold water
with severe stirring. The solution was extracted with ethyl
acetate, separated the organic layer, washed with brine, dried with
Na2SO4 and distilled under reduced pressure to obtain the titled
compound as brown solid in 93 % yield. mp: 139-141 °C; 1H NMR (400
MHz, CDCl3): δ 2.40 (s, 3H, CH3), 2.57 (s, 1H, CH), 4.76 (d, J=2.3
Hz, 2H, OCH2), 6.16 (s, 1H, ArH), 6.92 (d, J = 2.4, 1H, ArH), 6.94
(s, 1H, ArH), 7.52 (d, J = 9.2 Hz, 1H, ArH). 13C NMR (100 MHz,
CDCl3): δ 18.7, 56.3, 76.6, 77.6, 102.3, 112.5, 112.8, 114.4,
125.7, 152.5, 155.1, 160.5, 161.2. LC-MS: Calculated 214.2,
Observed 215.2. Analysis calcd for C13H10O3: C, 72.89, H, 4.71, O,
22.41 %, found: C, 72.92, H, 4.70, O, 22.38 %.
General procedure for the synthesis of compounds (4a-t)
To the weighed quantity of intermediate 3 (1 equiv.) in
t-butanol/water (1:1), were added azide (1.3 equiv.), CuSO4.5H2O
(0.1 equiv.) and sodium ascorbate (0.3 equiv.) and the reaction
mixture was placed in the microwave and heated for 2–5 min. at 90
°C at 110 W power. The reaction mixture was quenched with water and
extracted with DCM, dried in Na2SO4 and distilled in reduced
pressure to obtain the crude product. The crude product was further
purified by column chromatography and eluted in varying polarities
to obtain the titled compounds 4a-t.
7-((1-(2-Methoxycyclopentyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one
(4a)
White solid: mp 168-170 °C; 1H NMR (400 MHz, DMSO-d6): δ
1.62-1.67 (m, 1H, CH), 1.76-1.85 (m, 2H, CH2), 1.96-2.09 (m, 2H,
CH2), 2.23-2.28 (m, 1H, CH), 2.49 (d, J=1.7 Hz, 3H, CH3), 3.18 (s,
3H, OCH3), 4.00-4.04 (m, 1H, OCH), 4.83-4.88 (m, 1H, NCH), 5.25 (s,
2H, OCH2), 6.22 (d, J=1.0 Hz, 1H, ArH), 7.02-7.05 (dd, J=8.8, 2.5
Hz, 1H, ArH), 7.15 (d, J=2.4 Hz, 1H, ArH), 7.70 (d, J=8.8 Hz, 1H,
ArH), 8.39 (s, 1H, ArH). 13C NMR (100 MHz, DMSO-d6): δ 18.4, 23.5,
26.7, 33.5, 63.5, 65.7, 69.0, 76.1, 111.6, 113.7, 116.1, 124.9,
128.5, 133.9, 146.6, 154.2, 157.0, 160.6, 160.6. LC-MS: Calculated
355.2, Observed 356.2. Analysis calcd for C19H21N3O4: C, 64.21, H,
5.96, N, 11.82 %, found: C, 64.26, H, 5.93, N, 11.81 %.
7-((1-(2-Hydroxycyclohexyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one
(4b).
White solid: mp 167-169 °C; 1H NMR (400 MHz, DMSO-d6): δ
1.82-1.89 (m, 5H, CH), 1.95-1.97 (m, 3H, CH), 2.49 (d, J=1.7 Hz,
3H, CH3), 3.71-3.73 (m, 1H, OCH), 4.21-4.24 (m, 1H, NCH), 4.95 (d,
J=5.9 Hz, 1H, OH), 5.23 (s, 2H, OCH2), 6.22 (d, J=0.9 Hz, 1H, ArH),
7.03-7.06 (dd, J=8.8, 2.4 Hz, 1H, ArH), 7.16 (d, J=2.4 Hz, 1H,
ArH), 7.70 (d, J=8.8 Hz, 1H, ArH), 8.25 (s, 1H, ArH). 13C NMR (100
MHz, DMSO-d6): δ 18.4, 23.4, 24.7, 26.8, 33.5, 61.8, 69.7, 77.1,
112.7, 114.5, 115.8, 125.8, 128.2, 134.0, 147.9, 154.6, 157.6,
160.7, 161.0. LC-MS: Calculated 355.2, Observed 356.2. Analysis
calcd for C19H21N3O4: C, 64.21, H, 5.96, N, 11.82 %, found: C,
64.25, H, 5.92, N, 11.81 %.
7-((1-(2-Hydroxycyclopentyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one
(4c).
White solid: mp 155-157 °C; 1H NMR (300 MHz, DMSO-d6): δ
1.96-2.02 (m, 3H, CH), 2.18-2.23 (m, 2H, CH2), 2.25-2.30 (m, 1H,
CH2), 2.49 (d, J=1.7 Hz, 3H, CH3), 4.19-4.27 (m, 1H, OCH),
4.58-4.66 (m, 1H, NCH), 5.23 (s, 2H, OCH2), 5.23 (d, J=3.45 Hz, 1H,
OH), 6.21 (s, 1H, ArH), 7.01-7.05 (dd, J=8.8, 2.5 Hz, 1H, ArH),
7.15 (d, J=2.5 Hz, 1H, ArH), 7.70 (d, J=8.8 Hz, 1H, ArH), 8.32 (s,
1H, ArH). 13C NMR (75 MHz, DMSO-d6): δ 18.4, 23.4, 26.8, 33.5,
63.8, 69.4, 76.2, 111.9, 113.8, 116.1, 125.8, 128.5, 133.9, 147.7,
154.7, 157.6, 160.8, 160.9. LC-MS: Calculated 341.2, Observed
342.2. Analysis calcd for C18H19N3O4: C, 63.33, H, 5.61, N, 12.31
%, found: C, 63.37, H, 5.59, N, 12.30 %.
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7-((1-(2-(2-Phenoxycyclohexyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one
(4d).
White solid: mp 193-195 °C; 1H NMR (400 MHz, DMSO-d6): δ
1.72-1.84 (m, 3H, CH), 1.89-1.93 (m, 2H, CH2), 1.99-2.03 (m, 1H,
CH), 2.08-2.14 (m, 2H, CH2), 2.46 (d, J=1.2 Hz, 3H, CH3), 3.78-3.84
(m, 1H, OCH), 4.28-4.37 (m, 1H, NCH), 5.28 (s, 2H, OCH2), 6.24 (d,
J=1.1 Hz, 1H, ArH), 7.04-7.07 (dd, J=8.2, 2.0 Hz, 1H, ArH),
7.09-7.18 (m, 3H, ArH), 7.17 (d, J=2.5 Hz, 1H, ArH), 7.26-7.41 (m,
2H, ArH), 7.67 (d, J=8.1 Hz, 1H, ArH), 8.39 (s, 1H, ArH). 13C NMR
(100 MHz, DMSO-d6): δ 18.4, 23.4, 24.7, 26.8, 33.5, 64.7, 70.0,
78.1, 112.7, 114.5, 115.8, 118.1, 118.6, 119.2, 125.8, 127.3,
128.2, 129.5, 134.0, 147.9, 154.6, 157.6, 161.8, 160.7, 160.9.
LC-MS: Calculated 431.2, Observed 432.2. Analysis calcd for
C25H25N3O4: C, 69.59, H, 5.84, N, 9.74 %, found: C, 69.64, H, 5.83,
N, 9.72 %.
7-((1-(2-(2-Phenoxycyclopentyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one
(4e).
White solid: mp 189-191 °C; 1H NMR (400 MHz, DMSO-d6): δ
1.62-1.69 (m, 1H, CH), 1.74-1.86 (m, 2H, CH2), 1.95-2.02 (m, 2H,
CH2), 2.17-2.28 (m, 1H, CH), 2.40 (d, J=1.1 Hz, 3H, CH3), 4.27 (q,
J=1.6 Hz, 1H, OCH), 4.63 (dt, J=2.3, 1.4 Hz, 1H, NCH), 5.26 (s, 2H,
OCH2), 6.28 (d, J=1.2 Hz, 1H, ArH), 7.02-7.04 (dd, J=8.3, 2.1 Hz,
1H, ArH), 7.10-7.19 (m, 3H, ArH), 7.16 (d, J=2.4 Hz, 1H, ArH),
7.27-7.40 (m, 2H, ArH), 7.68 (d, J=8.2 Hz, 1H, ArH), 8.31 (s, 1H,
ArH). 13C NMR (100 MHz, DMSO-d6): δ 18.4, 23.6, 26.8, 33.5, 63.8,
69.7, 75.9, 111.7, 114.9, 116.2, 119.7, 124.8, 125.8, 126.3, 127.7,
128.6, 129.9, 133.8, 147.6, 154.7, 157.4, 159.9, 160.8, 161.2.
LC-MS: Calculated 417.2, Observed 418.2. Analysis calcd for
C24H23N3O4: C, 69.05, H, 5.55, N, 10.07 %, found: C, 69.08, H,
5.55, N, 10.06 %.
7-((1-(2-(2-Chloropyridin-3-yloxy)cyclohexyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one
(4f).
Light yellow solid: mp 180-182 °C; 1H NMR (400 MHz, DMSO-d6): δ
1.70-1.81 (m, 3H, CH), 1.85-1.90 (m, 2H, CH2), 1.95-2.01 (m, 1H,
CH), 2.08-2.16 (m, 2H, CH2), 2.45 (d, J=1.1 Hz, 3H, CH3), 3.75-3.82
(m, 1H, OCH), 4.27-4.33 (m, 1H, NCH), 5.27 (s, 2H, OCH2), 6.26 (d,
J=1.2 Hz, 1H, ArH), 7.04-7.07 (dd, J=8.2, 1.6 Hz, 1H, ArH), 7.17
(d, J=2.3 Hz, 1H, ArH), 7.41 (d, J=8.0 Hz, 1H, ArH), 7.67 (d, J=8.3
Hz, 1H, ArH), 8.13 (d, J=8.4 Hz, 1H, ArH), 8.39 (s, 1H, ArH), 8.67
(d, J=8.5 Hz, 1H, ArH). 13C NMR (100 MHz, DMSO-d6): δ 18.4, 23.5,
24.6, 25.9, 32.9, 64.6, 69.0, 77.7, 112.5, 114.4, 115.9, 125.8,
127.4, 128.9, 129.6, 133.8, 145.5, 146.5, 147.9, 154.6, 157.6,
159.8, 160.8, 161.2. LC-MS: Calculated 466.2, Observed 467.2.
Analysis calcd for C24H23ClN4O4: C, 61.74, H, 4.97, N, 12.00 %,
found: C, 61.78, H, 4.95, N, 11.99 %.
7-((1-(2-(2-Chloropyridin-3-yloxy)cyclopentyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one
(4g).
Light yellow solid: mp 203-205 °C; 1H NMR (400 MHz, DMSO-d6): δ
1.90-1.97 (m, 3H, CH), 2.33-2.38 (m, 3H, CH), 2.49 (d, J=1.7 Hz,
3H, CH3), 5.18-5.22 (m, 2H, NCH, OCH), 5.25 (s, 2H, OCH2), 6.21 (d,
J=1.0 Hz, 1H, ArH), 7.00-7.03 (dd, J=8.8, 2.4 Hz, 1H, ArH), 7.13
(d, J=2.4 Hz, 1H, ArH), 7.29-7.32 (m, 1H, ArH), 7.47-7.49 (dd,
J=8.2, 1.3 Hz, 1H, ArH), 7.68 (d, J=8.8 Hz, 1H, ArH), 7.95-7.96
(dd, J=4.6, 1.4 Hz, 1H, ArH), 8.41 (s, 1H, ArH). 13C NMR (100 MHz,
DMSO-d6): δ 18.4, 23.6, 26.8, 33.5, 63.8, 69.7, 75.9, 111.7, 114.9,
116.2, 126.3, 127.9, 128.6, 129.6, 133.8, 145.7, 147.6, 149.6,
154.7, 157.7, 159.9, 160.9, 161.2. LC-MS: Calculated 452.0,
Observed 453.0. Analysis calcd for C23H21ClN4O4: C, 61.00, H, 4.67,
N, 12.37 %, found: C, 61.03, H, 4.65, N 12.35 %.
7-((1-(4-Hydroxypyrrolidin-3-yl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one
(4h).
Yellow solid: mp 146-148 °C; 1H NMR (400 MHz, DMSO-d6): δ 2.39
(d, J=1.2 Hz, 3H, CH3), 3.55-3.76 (m, 1H, CH), 3.79-3.89 (m, 1H,
CH), 3.92-4.00 (m, 2H, CH2), 5.24 (d, J=1.8 Hz, 1H, OCH), 5.31 (s,
2H, OCH2), 5.39 (d, J=1.0 Hz, 1H, NCH), 5.60 (d, J=1.2 Hz, 1H, OH),
6.23 (d, J=1.0 Hz, 1H, ArH), 7.03-7.06 (dd, J=8.2, 1.5 Hz, 1H,
ArH), 7.16 (d, J=2.4 Hz, 1H, ArH), 7.70 (d, J=8.2 Hz, 1H, ArH),
8.55 (s, 1H, ArH), 9.72 (bs, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ
18.8, 44.3, 53.2, 64.8, 69.4, 77.4, 111.9, 113.7, 116.3, 125.7,
129.4, 133.6, 146.9, 154.7, 157.6, 160.6, 160.8. LC-MS: Calculated
342.0, Observed 343.0. Analysis calcd for C17H18N4O4: C, 59.64, H,
5.30, N, 16.37 %, found: C, 59.71, H, 5.26, N, 16.36 %.
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7-((1-(4-Phenoxypyrrolidin-3-yl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one
(4i).
Light yellow solid: mp 190-192 °C; 1H NMR (400 MHz, DMSO-d6): δ
2.49 (d, J=1.6 Hz, 3H, CH3), 3.55-3.60 (m, 1H, CH), 3.76-3.79 (m,
1H, CH), 3.89-3.96 (m, 2H, CH), 5.31 (s, 2H, OCH2), 5.40 (d, J=4.0
Hz, 1H, OCH), 5.61 (d, J=4.8 Hz, 1H, NCH), 6.23 (d, J=0.9 Hz, 1H,
ArH), 7.00-7.05 (m, 4H, ArH), 7.17 (d, J=2.4 Hz, 1H, ArH),
7.32-7.36 (m, 2H, ArH), 7.71 (d, J=8.8 Hz, 1H, ArH), 8.55 (s, 1H,
ArH), 9.72 (bs, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 18.8, 44.3,
53.2, 64.8, 69.4, 77.4, 111.9, 113.7, 116.3, 119.2, 120.3, 122.5,
125.7, 127.8, 128.6, 129.4, 133.6, 146.9, 154.7, 157.6, 158.8,
159.9, 160.8. LC-MS: Calculated 418.2, Observed 419.2. Analysis
calcd for C23H22N4O4: C, 66.02, H, 5.30, N, 13.39 %, found: C,
66.05, H, 5.29, N, 13.39 %.
7-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one
[32] (4j).
White solid: mp 118-120 °C; 1H NMR (400 MHz, DMSO-d6): δ 2.49
(d, J=1.2 Hz, 3H, CH3), 4.98 (s, 2H, NCH2), 5.25 (s, 2H, OCH2),
6.24 (d, J=1.2 Hz, 1H, ArH), 7.09-7.11 (dd, J=8.2, 1.6 Hz, 1H,
ArH), 7.16-7.19 (dd, J=8.6, 2.1 Hz, 2H, ArH), 7.21 (d, J=2.2 Hz,
1H, ArH), 7.36 (m, 1H, ArH), 7.69-7.71 (dd, J=8.7, 1.92 Hz, 2H,
ArH), 7.73 (d, J=7.3 Hz, 1H, ArH), 8.47 (s, 1H, ArH. 13C NMR (100
MHz, DMSO-d6): δ 18.8, 59.5, 76.8, 112.4, 114.5, 116.5, 125.8,
126.7, 128.6, 129.1, 131.6, 134.6, 143.5, 148.9, 154.5, 156.9,
159.3, 160.5. LC-MS: Calculated 347.0, Observed 348.0. Analysis
calcd for C20H17N3O3: C, 69.15, H, 4.93, N, 12.10 %, found: C,
69.20, H, 4.91, N, 12.09 %.
7-((1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one
(4k).
Off white solid: mp 175-177 °C; 1H NMR (400 MHz, DMSO-d6): δ
2.47 (d, J=1.1 Hz, 3H, CH3), 4.97 (s, 2H, NCH2), 5.24 (s, 2H,
OCH2), 6.22 (d, J=1.0 Hz, 1H, ArH), 7.01-7.04 (dd, J=7.8, 1.8 Hz,
2H, ArH), 7.11-7.14 (dd, J1=7.9, 1.9 Hz, 1H, ArH), 7.21 (d, J=2.5
Hz, 1H, ArH), 7.55-7.57 (dd, J=8.2, 2.2 Hz, 2H, ArH), 7.70 (d,
J=7.4 Hz, 1H, ArH), 8.51 (s, 1H, ArH). 13C NMR (100 MHz, DMSO-d6):
δ 18.8, 59.5, 76.8, 112.4, 114.5, 115.5 & 115.7 (d, 2JCF =21
Hz), 116.5, 125.8, 128.6, 129.7 & 129.8 (d, 3JCF =8 Hz), 134.6,
142.8, 148.9, 156.9, 158.3 & 160.7 (d, 1JCF =241.70 Hz), 161.3,
162.5. LC-MS: Calculated 365.0, Observed 366.0. Analysis calcd for
C20H17N3O3: C, 65.75, H, 4.41, N, 11.50 %, found: C, 65.79, H,
4.38, N, 11.51 %.
7-((1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one
[33] (4l).
White solid: mp 162-163 °C; 1H NMR (400 MHz, DMSO-d6): δ 2.46
(d, J=1.2 Hz, 3H, CH3), 4.95 (s, 2H, NCH2), 5.21 (s, 2H, OCH2),
6.24 (d, J=1.3 Hz, 1H, ArH), 7.03 (dd, J=7.6, 1.4 Hz, 1H, ArH),
7.10-7.13 (dd, J=7.8, 1.8 Hz, 2H, ArH), 7.29 (d, J=2.6 Hz, 1H,
ArH), 7.48-7.51 (dd, J=8.1, 2.1 Hz, 2H, ArH), 7.73 (d, J=7.6 Hz,
1H, ArH), 8.43 (s, 1H, ArH). 13C NMR (100 MHz, DMSO-d6): δ 18.7,
59.7, 76.8, 112.4, 114.5, 116.5, 125.8, 128.6, 129.0, 131.3, 134.6,
138.8, 143.7, 148.9, 154.5, 155.7, 158.2, 160.4. LC-MS: Calculated
381.1, Observed 382.1. Analysis calcd for C20H16ClN3O3: C, 62.91,
H, 4.22, N, 11.01 %, found: C, 62.95, H, 4.21, N, 10.99 %.
4-((4-((4-Methyl-2-oxo-2H-chromen-7-yloxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)benzonitrile
(4m).
White solid: mp 175-177 °C; 1H NMR (400 MHz, DMSO-d6): δ 2.51
(d, J=1.1 Hz, 3H, CH3), 4.99 (s, 2H, NCH2), 5.29 (s, 2H, OCH2),
6.33 (d, J=1.4 Hz, 1H, ArH), 7.01-7.04 (dd, J=7.6, 1.2 Hz, 1H,
ArH), 7.09-7.12 (dd, J=8.0, 2.0 Hz, 2H, ArH), 7.28 (d, J=2.7 Hz,
1H, ArH), 7.41-7.44 (dd, J=8.4, 2.0 Hz, 2H, ArH), 7.77 (d, J=7.6
Hz, 1H, ArH), 8.50 (s, 1H, ArH). 13C NMR (100 MHz, DMSO-d6): δ
18.7, 58.9, 76.8, 110.2, 112.4, 114.5, 116.5, 118.2, 125.8, 128.6,
128.8, 130.6, 134.6, 142.9, 148.9, 154.5, 156.8, 158.6, 161.1.
LC-MS: Calculated 372.1, Observed 373.1. Analysis calcd for
C21H16N4O3: C, 67.73, H, 4.33, N, 15.05 %, found: C, 67.78, H,
4.31, N, 15.04 %.
7-((1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one
[32] (4n).
Light yellow solid: mp 144-146°C; 1H NMR (400 MHz, DMSO-d6): δ
2.49 (d, J=1.4 Hz, 3H, CH3), 4.97 (s, 2H, NCH2), 5.26 (s, 2H,
OCH2), 6.21 (d, J=1.5 Hz, 1H, ArH), 6.96-6.99 (dd, J=7.4, 1.3 Hz,
1H, ArH), 7.07-7.09 (dd, J=8.0, 1.8 Hz, 2H, ArH), 7.26 (d, J=2.8
Hz, 1H, ArH), 7.37-7.40 (dd, J=8.1, 1.9 Hz, 2H, ArH), 7.68 (d,
J=7.5 Hz, 1H, ArH), 8.46 (s, 1H, ArH). 13C NMR (100 MHz, DMSO-d6):
δ 18.7, 59.4, 76.8, 112.4, 114.5, 116.5, 124.9 (2 peaks), 125.8,
128.6, 130.8 (2 peaks), 134.6, 142.9, 146.8, 148.9, 154.5, 156.8,
158.5,
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160.9. LC-MS: Calculated 392.1, Observed 393.1. Analysis calcd
for C20H16N4O5: C, 61.22, H, 4.11, N, 14.28 %, found: C, 61.27, H,
4.08, N, 14.26 %.
7-((1-(4-Hydroxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one
(4o).
Light brown solid: mp 144-146 °C; 1H NMR (400 MHz, DMSO-d6): δ
2.46 (d, J=1.1 Hz, 3H, CH3), 4.93 (s, 2H, NCH2), 5.19 (s, 2H,
OCH2), 6.24 (d, J=1.3 Hz, 1H, ArH), 6.94-6.97 (dd, J=7.3, 1.4 Hz,
1H, ArH), 7.07-7.10 (dd, J=7.6, 1.5 Hz, 2H, ArH), 7.28 (d, J=2.4
Hz, 1H, ArH), 7.36-7.39 (dd, J=8.0, 1.9 Hz, 2H, ArH), 7.68 (d,
J=7.4 Hz, 1H, ArH), 8.57 (s, 1H, ArH), 10.67 (bs, 1H, OH). 13C NMR
(100 MHz, DMSO-d6): δ 18.8, 59.4, 76.8, 112.4, 114.5, 116.5, 117.5
(2 peaks), 125.8, 128.6, 131.1 (2 peaks), 133.4, 134.6, 148.9,
154.5, 155.8, 157.0, 160.3, 161.0. LC-MS: Calculated 363.1,
Observed 364.1. Analysis calcd for C20H17N3O4: C, 66.11, H, 4.72,
N, 11.56 %, found: C, 66.17, H, 4.69, N, 11.55 %.
7-((1-(4-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one
(4p).
Off white solid: mp 178-180 °C; 1H NMR (400 MHz, DMSO-d6): δ
2.51 (d, J=1.0 Hz, 3H, CH3), 3.45 (s, 3H, OCH3), 4.99 (s, 2H,
NCH2), 5.29 (s, 2H, OCH2), 6.33 (d, J=1.3 Hz, 1H, ArH), 7.01-7.04
(dd, J=7.6, 1.7 Hz, 1H, ArH), 7.09-7.12 (dd, J=7.8, 2.0 Hz, 2H,
ArH), 7.28 (d, J=2.2 Hz, 1H, ArH), 7.41-7.43 (dd, J=8.1, 2.0 Hz,
2H, ArH), 7.77 (d, J=7.4 Hz, 1H, ArH), 8.50 (s, 1H, ArH). 13C NMR
(100 MHz, DMSO-d6): δ 18.7, 58.5, 66.6, 76.9, 112.9, 114.7, 116.0,
116.3 (2 peaks), 125.8, 128.6, 130.6 (2 peaks), 133.0, 134.3,
146.4, 148.7, 155.0, 157.0, 160.7, 161.0. LC-MS: Calculated 377.1,
Observed 378.1. Analysis calcd for C21H19N3O4: C, 66.83, H, 5.07,
N, 11.13 %, found: C, 66.87, H, 5.05, N, 11.11 %.
7-((1-(4-Aminobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one
(4q).
Yellow solid: mp 173-175 °C; 1H NMR (400 MHz, DMSO-d6): δ 2.46
(d, J=1.2 Hz, 3H, CH3), 4.95 (s, 2H, NCH2), 5.21 (s, 2H, OCH2),
5.88 (bs, 2H, NH2), 6.24 (d, J=1.4 Hz, 1H, ArH), 7.03-7.05 (dd,
J=7.2, 1.5 Hz, 1H, ArH), 7.10-7.13 (dd, J=7.5, 1.8 Hz, 2H, ArH),
7.29 (d, J=2.4 Hz, 1H, ArH), 7.48-7.51 (dd, J=7.8, 2.0 Hz, 2H,
ArH), 7.73 (d, J=7.8 Hz, 1H, ArH), 8.43 (s, 1H, ArH). 13C NMR (100
MHz, DMSO-d6): δ 18.8, 59.4, 76.8, 112.4, 114.5, 116.5, 117.5 (2
peaks), 125.8, 128.6, 129.8, 131.1 (2 peaks), 134.6, 148.9, 154.5,
155.8, 157.0, 160.3, 161.0. LC-MS: Calculated 362.1, Observed
363.1. Analysis calcd for C20H18N4O3: C, 66.29, H, 5.01, N, 15.46
%, found: C, 66.32, H, 5.00, N, 15.44 %.
7-((1-(4-(Methylamino)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one
(4r).
Off white solid: mp 177-179 °C; 1H NMR (400 MHz, DMSO-d6): δ
2.51 (d, J=1.2 Hz, 3H, CH3), 3.02 (s, 3H, NCH3), 4.99 (s, 2H,
NCH2), 5.29 (s, 2H, OCH2), 6.33 (d, J=1.2 Hz, 1H, ArH), 7.01-7.04
(dd, J=7.2, 1.3 Hz, 1H, ArH), 7.09-12 (dd, J=7.5, 1.8 Hz, 2H, ArH),
7.28 (d, J=2.7 Hz, 1H, ArH), 7.41-7.43 (dd, J=8.1, 2.2 Hz, 2H,
ArH), 7.77 (d, J=7.4 Hz, 1H, ArH), 7.84 (bs, 1H, NH), 8.53 (s, 1H,
ArH). 13C NMR (100 MHz, DMSO-d6): δ 18.6, 38.4, 59.1, 76.2, 111.9,
114.3, 116.1, 117.5 (2 peaks), 126.2, 128.2, 129.7, 130.8 (2
peaks), 134.1, 147.9, 154.1, 155.7, 156.9, 159.1, 160.9. LC-MS:
Calculated 376.2, Observed 377.2. Analysis calcd for C21H20N4O3: C,
67.01, H, 5.36, N, 14.88 %, found: C, 67.06, H, 5.34, N, 14.87 %.
4-Methyl-7-((1-(4-methyl-benzyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one
(4s).
White solid: mp 171-173 °C; 1H NMR (400 MHz, DMSO-d6): δ 2.33
(s, 3H, CH3), 2.46 (d, J=1.4 Hz, 3H, CH3), 4.93 (s, 2H, NCH2), 5.19
(s, 2H, OCH2), 6.24 (d, J=1.3 Hz, 1H, ArH), 6.94-6.97 (dd, J=7.5,
1.3 Hz, 1H, ArH), 7.07-7.09 (dd, J=7.8, 1.7 Hz, 2H, ArH), 7.28 (d,
J=2.5 Hz, 1H, ArH), 7.36-7.39 (dd, J=8.1, 1.88 Hz, 2H, ArH), 7.68
(d, J=7.4 Hz, 1H, ArH), 8.57 (s, 1H, ArH). 13C NMR (100 MHz,
DMSO-d6): δ 18.3, 26.8, 58.7, 76.8, 112.4, 114.5, 116.5, 125.8,
128.1, 128.6 (2 peaks), 130.1 (2 peaks), 134.6, 135.4, 136.3,
148.9, 154.5, 156.9, 159.3, 160.5. LC-MS: Calculated 361.0,
Observed 362.0. Analysis calcd for C21H19N3O3: C, 69.79, H, 5.30,
N, 11.63 %, found: C, 69.84, H, 5.29, N, 11.61 %.
7-((1-(4-Ethylbenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-methyl-2H-chromen-2-one
(4t).
White solid: mp 175-177 °C; 1H NMR (400 MHz, DMSO-d6): δ 2.31
(t, J=4.5 Hz, 3H, CH3), 2.44 (d, J=1.2 Hz, 3H, CH3), 4.90 (s, 2H,
NCH2), 5.06 (q, J=4.7 Hz, 2H, CH2), 5.19 (s, 2H, OCH2), 6.24 (d,
J=1.3 Hz,
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1H, ArH), 6.90-6.93 (dd, J=7.3, 1.2 Hz, 1H, ArH), 7.05-7.08 (dd,
J=7.7, 1.6 Hz, 2H, ArH), 7.21 (d, J=2.6 Hz, 1H, ArH), 7.37-7.40
(dd, J=8.0, 1.8 Hz, 2H, ArH), 7.66 (d, J=7.5 Hz, 1H, ArH), 8.53 (s,
1H, ArH). 13C NMR (100 MHz, DMSO-d6): δ 18.9, 19.0, 31.1, 58.1,
76.6, 112.3, 115.3, 116.4, 126.1, 128.3, 128.4 (2 peaks), 129.9 (2
peaks), 133.9, 135.0, 135.8, 148.0, 154.3, 155.8, 159.7, 160.9.
LC-MS: Calculated 375.2, Observed 376.2. Analysis calcd for
C22H21N3O3: C, 70.38, H, 5.64, N, 11.19 %, found: C, 70.43, H,
5.63, N, 11.16 %.
Biology
The experimental procedure for the determination of biological
activities is detailed in the Supporting Information. In silico
studies
An entirely in-house developed drug discovery informatics system
OSIRIS was used to perform ADMET based calculations. It is a Java
based library layer that provides reusable cheminformatics
functionality and was used to predict the toxicity risks and
overall drug score via in silico [52]. The structure of synthesized
molecules and the standards were drawn in ChemBioDraw tool
(ChemBioOffice Ultra 14.0 suite) assigned with proper 2D
orientation and structure of each one was checked for structural
drawing error. Energy of each molecule was minimized using
ChemBio3D (ChemBioOffice Ultra 14.0 suite). The energy minimized
ligand molecules were then used as input for AutoDockVina, in order
to carry out the docking simulation [53]. The protein databank
(PDB) coordinate file entitled ‘2XCT.pdb’ was used as receptor
(protein) molecule which is a structure of S. aureus gyrase in
complex with Ciprofloxacin and DNA [54]. All the water molecules
were removed from the receptor and SPDBV DeepView was used to
automatically rebuild the missing side chains in the receptor. The
Graphical User Interface program ‘MGL Tools’ was used to set the
grid box for docking simulations. The grid was set so that it
surrounds the region of interest (active site) in the
macromolecule.
In the present study, the active site was selected based on the
amino acid residues of 2XCT, which are involved in binding with
Ciprofloxacin. Therefore, the grid was centered at the region
including the 2 amino acid residues (Arg 458 and Gly 459) and 4
nitrogenous bases from DNA that is guanine (G), adenine (A),
thymine (T) or cytosine (C) as evidenced by the work of Bax et al.,
2010 [55]. This surrounds the active site. The grid box volume was
set to 8, 14, and 14 Å for x, y and z dimensions respectively, and
the grid center was set to 3.194, 43.143 and 69.977 for x, y and z
center respectively, which covered the 2 amino acid residues and 4
nitrogenous bases in the considered active pocket. AutoGrid 4.0
Program supplied with AutoDock 4.0 was used to produce grid maps
[55]. The docking algorithm provided with AutoDockVina was used to
search for the best docked conformation between ligand and protein.
During the docking process, a maximum of 100 conformers was
considered for each ligand. All the AutoDock docking runs were
performed in Core i7 Intel processor CPU with 8 GB DDR3l RAM.
AutoDockVina was compiled and run under Windows 8.0 professional
operating system. LigPlot+ [56] and PyMol [51] were used to deduce
the pictorial representation of interaction between the ligands and
the target protein.
Results and discussion Chemistry
As depicted in Schemes 1 and 2, we started our synthetic
strategy by the synthesis of parent 4-methyl-7-hydroxy coumarin 2
by the modified Pechmann cyclization reaction in which resorcinol 1
was treated with ethyl acetoacetate in 1-butyl-3-methylimidazolium
chloro aluminate at 30 °C for 20 minutes [36]. The obtained
4-methyl-7-hydroxy coumarin intermediate 2 was then treated with
propargyl bromide in K2CO3 to procure the O-propargylated product
3. The alkyne intermediate 3 thus obtained was further subjected to
the copper catalyzed 1,3 dipolar cycloaddition reaction with
various azides under microwave irradiation at 90 °C with the
intention of synthesizing an array of coumarin derivatives with
potent antimicrobial and antioxidant properties.
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OHO OBr
K2CO3O OO
32
[bmim]Cl.2AlCl3CH3COCH2COOEt
HO OH
1 30oC, 20 min.
AcetoneRT, 12h.
Scheme 1. Synthesis of 4-methyl-7-propargylated coumarin
intermediate.
RN3
Na-AscorbateCuSO4.5H2O
OO O
4a-t
NN
N
R
Microwave, 90oC
O OO
3
t-BuOH-H2O (1:1)
2-5 min. Scheme 2. Synthesis of coumarins linked with
1,2,3-triazoles.
As a model substrate, we started our initial screening by
treating the alkyne intermediate 3 with 2-methoxy cyclopentyl azide
in sodium ascorbate and hydrated copper sulfate (CuSO4.5H2O) at 90
°C in microwave. The solvent system used for the reaction
optimization was an equimolar mixture of t-butanol and water. To
our delight, we identified the reaction completion within 5 min. by
TLC and further analysis and purification procured the expected
product in 100 % yield (LC-MS) with 97 % isolated yield. In order
to validate the predominance of microwave irradiation, we carried
out the same reaction at room temperature as well as standard
thermal conditions (Table 1, Entry 1). The reaction took 6 h. for
completion under conventional heating with 80 % yield while it
required 18 h. under ambient temperature and the yield of the
product was found to be 86 %.
With the promising results in hand, our next attention was to
explore the generality of this synthetic methodology. Keeping this
in mind, we treated the intermediate 3 with a series of aliphatic
azides under microwave irradiation. Gratifyingly, all the azides
reacted efficiently to render the 1,2,3-triazoles linked with
coumarins in excellent yields. We also extended our developed
methodology for the synthesis of 1,2,3-triazoles with different
benzylic substituents linked with coumarins (Table 1, Entries
10-20). To our delight, all the reactions furnished the required
products in good to excellent yields. Microwave irradiation proved
to be superior in terms of yield as well as reaction time when
compared to other standard conditions (Table 1, Entries 1, 2 and
10).
Table 1. Click chemistry reaction of alkyne intermediate 3 with
various azides. Entry Azide Product (4a-t) Time Yieldb
(%) 1
N3
O
OO O
NNN
O
4a
3 min 6 hc
18 hd
97 80 86
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2 OHN3
OO O
NNN
OH
4b
3 min 6 hc
18 hd
95 76 85
3 N3
OH
OO O
NNN
OH
4c
3 min
94
4
ON3
OO O
NNN
O
4d
4 min
92
5 O
N3
OO O
NNN
O
4e
4 min
90
6
ON
ClN3
OO O
NNN
ON
Cl
4f
4 min 93
7 O N
ClN3
OO O
NNN
ON
Cl
4g
4 min 94
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8
NH
OHN3
OO O
NNN
NH
OH
4h
3 min 97
9
NH
ON3
OO O
NNN
NH
O
4i
4 min 90
10 N3
OO O
NNN
4j
3 min 6 hc
18 hd
98 83 87
11 N3
F
OO O
NNN
F 4k
3 min
95
12 N3
Cl
OO O
NNN
Cl 4l
3 min
93
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13 N3
NC
OO O
NNN
NC 4m
4 min
92
14 N3
O2N
OO O
NNN
O2N 4n
5 min 90
15 N3
HO
OO O
NNN
HO 4o
3 min
96
16 N3
O
OO O
NNN
O 4p
3 min
95
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17 N3
H2N
OO O
NNN
H2N 4q
3 min
98
18 N3
NH
OO O
NNN
NH 4r
3 min
98
19 N3
OO O
NNN
4s
3 min
96
20 N3
OO O
NNN
4t
3 min
95
a Reaction conditions: Alkyne 3 (1 equiv.), Azide (1.3 equiv.),
CuSO4.5H2O (0.1 equiv.), Sodium ascorbate (0.3 equiv.), t-BuOH/H2O
(1:1), microwave irradiated at 90 °C. b Isolated yield. c Reaction
carried out by conventional heating. d Reaction carried out at room
temperature.
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Biology Antimicrobial activity
The clinical relevance of bacterial and fungal diseases has
increased over the past 30 years due to an increasing population of
immunocompromised patients who suffer from various illnesses. The
development of multi drug resistance among pathogens could be a
major reason for this increasing issue [37]. In view of these facts
and stimulated by the profound activity profile of coumarins and
1,2,3-triazoles, we carried out the analysis of antibacterial and
antifungal activities of the newly synthesized compounds 4a-t
against two Gram-positive bacteria (Staphylococcus aureus ATCC
25923 and Bacillus subtilis ATCC 6633), two Gram-negative bacteria
(Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922)
and three fungi (Aspergillus flavus ATCC 9643, Chrysosporium
keratinophilum ATCC90272 and Candida albicans MTCC 227). The
results from the evaluation of antimicrobial activities in mg/mL
concentration are illustrated in Table 1 and Table 2 of the
Supplementary Information. Some of the tested compounds showed
promising antibacterial activity when compared to the standard
drug, Ciprofloxacin (See Table 1, Supplementary Information). The
compounds 4f, 4g, 4k, 4l, 4m and 4s showed promising activity when
compared with the standard while the compounds 4b, 4c, 4d, 4j, 4o
and 4r failed to show any activity against the tested strains. All
the other compounds displayed moderate to poor antibacterial
activity.
The minimum inhibitory concentration (MIC) of the more active
compounds was determined by broth dilution method (Table 2). From
the results, it was acknowledged that S. aureus (5 µg MIC) was the
most susceptible, and E. coli (10 µg MIC), P. aeruginosa (10 µg
MIC) and B. subtilis (10 µg MIC) were the most insensitive strain
among all the bacteria used in this study. The compound 4k was
found to be active against all the bacterial strains. The 4a, 4i,
4p (150 µg/mL MIC) and 4t (150 µg/mL MIC) showed weak activity as
compared to 4f (10 µg/mL MIC), 4g (10 µg/mL MIC), 4k (10 µg/mL
MIC), 4l (100 µg/mL MIC), 4m (100 µg/mL MIC) and 4s (125 µg/mL MIC)
against E.coli (Table 2). In the last years, Gram-negative bacteria
are frequently being reported to have developed multi drug
resistance to many of the antibiotics that are currently available
in the market of which E. coli is the most prominent [38,39]. This
could be the plausible reason for the high MIC values for E. coli
as compared to S. aureus.
Table 2. Minimum inhibitory concentration of synthesized
compounds for antibacterial activity.
Compounds in µg/mL
Escherichiacoli Staphylococcusaureus Pseudomonasaeruginosa
Bacillussubtilis
4a --- 100 100 50 4f 10 10 10 25 4g 10 10 10 10 4i --- 100 50
100 4k 10 5 10 10 4l 100 100 25 50
4m 100 75 10 25 4p 150 100 75 50 4s 125 --- 75 50 4t 150 100 75
100
Ciprofloxacin 0.6 0.2 0.5 0.4
The antifungal activity of the newly synthesized compounds was
initially carried out in mill molar
concentrations by taking Fluconazole as the standard (See Table
2, Supplementary Information). Among the 20 synthesized organic
compounds, only a few compounds inhibited the growth of most of the
human pathogenic fungi tested. The fungal sensitivity varied
according to the tested species. The compounds showed similar
antifungal activities to one another. The minimum inhibitory
concentration values for the antifungal activity of more active
compounds are summarized in Table 3. Among the tested fungi, C.
albicans was less sensitive when compared to the other fungal
species. A. flavus and C. keratinophilm showed some differing
responses to each organic compound. The compound 4n showed good
activity when compared to the remaining compounds. On the other
hand, all the newly synthesized organic compounds failed to show
good and comparable activity
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to that of the standard. The compounds 4b, 4c, 4d, 4e, 4h, 4j,
4o and 4r failed to show any activity towards the panel of fungal
pathogens.
Table 3. Minimum inhibitory concentration of synthesized
compounds for antifungal activity.
Compounds in µg/mL
Aspergillus flavus Chrysosporium keratinophilum Candida
albicans
4a 300 350 400 4f 300 250 350 4g >500 >500 >500 4i
>500 >500 >500 4k 250 200 200 4l >500 >500
>500
4m >500 >500 >500 4n 200 150 150 4p >500 >500
>500 4q >500 >500 >500 4s 300 250 350 4t 350 300
400
Fluconazole 10 20 30
Structure-activity relationships
The presence of electron withdrawing fluoro group in 4k is
presumed to be the sole reason for the comparable antibacterial
activity of that compound. The presence of heterocyclic ring having
a chloro substituent was assumed to be beneficial for the enhanced
activity of compounds 4f and 4g. The electron withdrawing groups
are expected to increase the lipophilicity and thereby enhance the
cell permeability of the molecule and hence improved its potency
[40]. In general, it can be summarized that in the present study,
the presence of ring substitution with an electron withdrawing
group and heterocyclic group at position 1 of 1,2,3-triazoles
linked with coumarins is an essential feature for the antimicrobial
effect of the synthesized compounds. Antioxidant activity
The DPPH procedure is one of the most effective methods for
evaluating the concentration of radical scavenging materials as it
does not have to be generated prior to analysis [41]. DPPH radical
scavenging activity evaluation is a rapid and convenient assay for
screening the antioxidant activities of products and has been
successfully applied for the evaluation of radical scavenging
activity of newly synthesized coumarin derivatives [42] as they
possess an extended p-conjugated system. Owing to these
observations, we directed our work towards the evaluation of
antioxidant activity of the synthesized compounds 4a-t by DPPH
assay. Table 4. Determination of antioxidant activity of the
synthesized compounds.
Entry Compound % Inhibition at 100 µg Concentration 1 4a 54.2 2
4b 66.1 3 4c 68.3 4 4d 36.4 5 4e 31.4 6 4f 26.1 7 4g -- 8 4h 74.2 9
4i 41.8 10 4j -- 11 4k --
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12 4l 28.1 13 4m 30.7 14 4n 56.3 15 4o 61.8 16 4p -- 17 4q 73.5
18 4r 29.4 19 4s -- 20 4t -- 21 Standard (BHT) 88.6
The synthesized compounds were subjected to antioxidant
screening by taking Butylated
hydroxytoluene (BHT) as the standard and our results are
summarized in Table 4. In this assay, the standard BHT showed a
strong scavenging activity, while the compounds 4b (66.1 %), 4h
(74.2 %), 4o (61.8 %), 4q (73.5 %) and 4c (68.3 %) displayed a
comparable activity (Fig 2). Unfortunately, the compounds 4g, 4j,
4k, 4p, 4s and 4t didn’t exhibit any activity when compared with
the standard and hence are considered as inactive. All the other
compounds also showed significant scavenging activity, but demanded
higher concentrations of the compounds.
Fig 2: The DPPH• scavenging activity of synthesized compounds in
comparison with the standard.
Structure-activity relationships The results of antioxidant
screening revealed that the presence of electron donating ring
systems
attached to the position 1 of 1,2,3-triazole ring linked with
coumarins is an indispensable characteristic for their radical
scavenging activity. The hydrophilic electron-donating groups are
expected to facilitate the stabilization of the oxygen-centered
radical and reduce the O–H bond dissociation enthalpy (BDE),
thereby increasing the radical scavenging activity by hydrogen
abstraction [43,44]. This could be the plausible reason for the
superior activity of compounds 4b, 4h, 4o, 4q and 4c to that of the
other synthesized molecules.
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Molecular docking studies The Gyrase enzyme relieves strain
while the double-stranded DNA is being unwound by helicase
[45,46]. It is an essential enzyme in all bacteria but absent in
higher eukaryotes, hence making it an interesting antibacterial
target [47-50]. Furthermore, the mode of antibacterial action of
Ciprofloxacin is by significantly inhibiting the gyrase enzyme.
Hence, the molecular docking studies of the active compounds with
gyrase were carried out and reported. Stimulated by the comparable
antibacterial activity of some of the synthesized compounds (4f,
4g, 4k and 4l) with the standard as per the in vivo results, it was
thought worthy to substantiate those results by performing the
molecular docking studies or in silico studies. The comparative
docking of receptor gyrase with 4f, 4g, 4k, 4l and the standard,
Ciprofloxacin, exhibited good affinity. They established hydrogen
bonding with one or more amino acids in the receptor active pocket
as represented in Table 5. Table 5. Binding affinity (kcal/mol),
H-bonds, H-bond length and H-bond formation of the standard and the
selected molecules after in silico docking.
Ligand Affinity (kcal/mol)
H-Bonds H-Bond Length
(Å)
H-Bond Between Hydrophobic interactions
4f -6.9 4 2.80 4f:N3::Glu435:OE2 Phe1123, Asp437, Gly436,
Gly459, Lys460, His1081
3.04 4f:N :: Gly1082:N 3.09 4f:O4 :: Ser1085:OG 3.14 4f:N2 ::
Glu435:O
4g -6.3 3 2.83 4g:N4::Glu435:OE2 Asp512, Ile516, His1081,
Gly1082, Arg1122, Phe1123, Gly436
2.96 4g:O3::Asp437:N 3.17 4g:O3::Ser438:N
4k -7.2 4 2.99 4k:O3::Ser1085:OG Ile516, Lys460, Gly436, Gly459,
Phe1123, His1081
3.12 4k:O3::Gly1082:N 3.12 4k:N2::Glu435:O 3.15
4k:E::Arg1122:NH1
4l -6.1 1 2.90 4l:O3::Gly459:N Arg458, Asp437, Gly436, Arg1122,
Phe1123, Glu435, Asp512, Asp510
Ciprofloxacin -6.2 2 2.83 Cipr:O3::Asp510:OD2 Gly459, Asp437,
Gly436, Phe1123, Asp512, 3.02 Cipr:O2::His1081:ND1
The 2D representation of the synthesized ligands 4f, 4g, 4k, 4l
and the standard Ciprofloxacin is
depicted in Figure 3. The compound 4k was found to be the best
of all the molecules taken under investigation as it possessed
significant hydrogen bonding as well as hydrophobic interactions.
For 4k (Fig 3), hydrophobic contacts were seen with six different
residues and four H-bonds were formed with various amino acids
(Table 5). The standard Ciprofloxacin (Fig 3) represented
hydrophobic contacts with five different residues, later a total of
two H-bonds were formed with various amino acids (Table 5). In all
the cases of the 2D representation, ligands are highlighted in
purple colour. The set of conserved residues that are commonly
involved in interaction with the ligands and Ciprofloxacin are
encircled with red colour.
http://en.wikipedia.org/wiki/DNA
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Fig 3. 2D representation of the interaction of 4f, 4g, 4k, 4l
and Ciprofloxacin with 2XCT (gyrase)
Based upon the obtained affinity, the best of the synthesized
ligands i.e., 4k along with the standard
Ciprofloxacin was subjected to 3D protein-ligand interaction
analysis. Figure 4 represents the further extrapolation of binding
conformation of 4k and Ciprofloxacin. Figures 4 (A) and (B)
represent the 3D interaction of 4k and Ciprofloxacin respectively
with gyrase by using educational version of PyMol [51]. The ligands
are represented in green colour, H-bonds with their respective
distances are represented with yellow colour, and the interacting
residues are represented in ball and stick model
representation.
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Fig 4. (A) 3D representation of the interaction of 4k and (B)
Ciprofloxacin with 2XCT (gyrase)
In the present study, 4k was identified to be the best
antibacterial agent among all the synthesized
compounds which could be attributed to the electron withdrawing
character of fluorine atom as well as the ability of the molecule
to form significant hydrogen bonding.
Conclusion
We have achieved a rapid, facile and efficient access for the
synthesis of an array of 1,2,3-triazoles linked with coumarins via
click chemistry and evaluated their antimicrobial and antioxidant
properties. Microwave irradiation proved to be superior to other
conventional methods for this synthetic methodology in terms of
yield as well as reaction time. The compounds 4k and 4g exhibited
promising antibacterial activity when compared with Ciprofloxacin
against all the tested bacteria. The in silico docking studies of
the more active antibacterials were carried out against the gyrase
enzyme and found that 4k possessed significant hydrogen bonding and
hydrophobic interactions which could also be the plausible reason
for its improved potency along with the presence of electron
withdrawing fluoro group. The compound 4n displayed better
antifungal activity when compared to other synthesized compounds
but were not promising when compared with the standard,
flucanazole. The compounds 4h and 4q showed comparable antioxidant
activity with the standard, BHT, presumably due to the presence of
electron donating substituents. The present study paved the way for
the synthesis of various coumarin analogues with significant
pharmacological properties and further derivatization and lead
optimization are in progress.
Acknowledgements The authors are thankful to the Department of
Industrial Chemistry, Kuvempu University for rendering
all the facilities to carry out the experiments. Vasiliy Bakulev
is thankful to Russian Foundation for Basic Research (Grant #
170300641A).
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